Here are our experiments & protocols

LB Preparation

• Liquid Medium with Chloramphenicol (CM) Preparation
  1. 8g of LB broth (Bioshop LBL407.1) are added with 400ml ddH₂O
  2. the solution is then sterilized
  3. after the cooldown, 200 μl CM is added
• Solid Medium with CM Preparation
  1. 8g of LB broth (Bioshop LBL407.1) are added with 400ml ddH₂O
  2. add 6g agar to the solution
  3. the solution is then sterilized
  4. after the cooldown, 200 μl CM is added
  5. pour the solution into the petri dish

Plasmid Extraction

• Preparation
  1. single colony is picked on the LB plate with CM
  2. the colony is cultured in 5ml LB+CM medium for 16-18hr
• Steps
  • Harvest
    1. transfer 1ml of cultured bacterial cells to a 1.5 ml microcentrifuge tube
    2. centrifuge at 14-16000 x g for 1 minute at room temperature
    3. discard the supernatant completely
    4. repeating the harvesting step in the same tube until 3 ml sample are collected
  • Resuspend
    1. add 200 μl of PD1 buffer (make sure Rnase A was added) to the Eppendorf
    2. resuspend the pellet by vortex or pipette until the cell pellet is completely dissolved
  • Cell Lysis
    1. add 200 μl of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times. (do not vortex, it will cause the shearing of the genomic DNA)
    2. stand the sample at room temperature for 2 to 5 minutes. Do not exceed 5 minutes
  • Neutralization
    1. add 300 μl of PD3 Buffer to the sample then mix immediately by inverting the tube 10 times
    2. centrifuge at 14-16000 x g for 3 minutes at room temperature
    3. during the centrifugation, place a PDH column in a 2 ml collection tube
  • DNA Binding
    1. transfer all of the supernatants to the PDH column
    2. centrifuge at 14-16000 x g for 30 seconds at room temperature
    3. the DNA binding step can be repeated for better yield
    4. discard the flow-through and place the PDH column back in the 2 ml Collection Tube
  • Wash
    1. add 400 μl W1 buffer to PDH column
    2. centrifuge at 14-16000 x g for 30 sec
    3. discard the flow-through and place the PDH column back into the collection tube
    4. add 600 μl of Wash buffer into the PDH column
    5. stand for 1 min at room temp, then centrifuge at 14-16000 x g for 30 sec
    6. discard the flow-through and place the PDH column back in the 2 ml collection tube
    7. centrifuge at 14-16000 x g for 3 min to dry the matrix
  • Elution
    1. add 400 μl of W1 buffer into the PDH column
    2. centrifuge at 14-16000 x g for 30 sec. Discard the flow-through and place the PDH column back into the collection tube
    3. add 600 μl of Wash buffer(make sure ethanol was added) into the PDH column
    4. let stand for 1 min at room temp. Centrifuge at 14-16000 x g for 30 sec then discard the flow-through
    5. place the PDH column back in the 2 ml collection tube
    6. centrifuge at 14-16000 x g for 3 min to dry the matrix

PCR

• Preparation

  • Component	Sample	Control
    10X KOD buffer	2	2
    MgSO4	0.8	0.8
    DNA template	60-80 ng (if using gDNA template, increase the amount of the template)	0
    dNTP (2mM)	0.5	0.5
    primer pair	1	1
    KOD plus enzyme	0.2-0.4	0.2-0.4
    add ddH2O to 20 μl

  • Component	Sample	Control
    2X dreamtaq green master mix	10 μl	10 μl
    DNA template	60-80 ng (if using gDNA template, increase the amount of the template)	0
    primer pair	1	1
    add ddH2O to 20 μl

• Machine set up

  • Step	Temp(°C)	Time
    1	94	5 min
    2	94	30 sec
    3	57-59	30 sec
    4	68-72	1 min for each kb
    5	68-72	7 min
    repeat step 3 to step 5 for 30-35 cycle
    6	20	10 min

Enzyme Digestion

• Preparation

  • component	amount per reaction
    DNA	1000 ng
    rCutsmart buffer	2 μl
    restriction enzyme	0.5 μl
    add sterilized ddH2O to 20 μl

  • component	amount per reaction
    DNA	1000 ng
    rCutsmart buffer	2 μl
    restriction enzyme 1	0.5 μl
    restriction enzyme 2	0.5 μl
    add sterilized ddH2O to 20 μl

• Steps
  1. prepare the sterilized eppendorf
  2. add the component above in the following order. ddH₂O, rCutsmart buffer, DNA, restriction enzyme
  3. mix the component by pipetting
  4. put it in the 37°C water bath for an hour

Electrophoresis

• Preparation
  • Large : 50ml TAE buffer + 0.5 - 1 g agarose (1-2%)
  • Small : 30 ml TAE buffer + 0.3 - 0.6 g agarose (1-2%)
• Steps
  1. add the component with protocol above
  2. heat the liquid by microwave oven to dissolve the agarose
  3. wait for it cool down until you can touch it with your hand and still feeling temperature
  4. pour agarose onto the gel tray
  5. add 4 μl loading dye to 20 μl sample for big well or add 2 μl loading dye to 10 μl sample for small well
  6. load sample and marker
  7. run 70 V, at least 30 min

Gel extraction

• Gel Dissociation
  1. prepare a 55-60°C dry bath
  2. transfer the gel slice to the eppendorf
  3. add 500 μl of gel buffer to the sample then mix by vortex (if two slice are used, added 600 μl instead)
  4. incubate at 55-60°C for 10-15 min to ensure the gel slice is completely dissolved
  5. during incubation, invert the tube every 2-3 min
• DNA binding
  1. place a DFH Column in a 2 ml Collection tube
  2. wait for the medium cool down to the room temperature
  3. transfer 800 μl of the sample to 2 ml Collection tube
  4. centrifuge at 14-16000 x g for 30 sec
  5. discard the flow-through and put the DFH column back in the 2 ml collection tube
• Wash
  1. add 400 μl W1 buffer to DFH column
  2. centrifuge at 14-16000 x g for 30 sec
  3. discard the flow-through and place the DFH column back into the collection tube
  4. add 600 μl of Wash buffer into the DFH column
  5. stand for 1 min at room temp, then centrifuge at 14-16000 x g for 30 sec
  6. discard the flow-through and place the DFH column back in the 2 ml collection tube
  7. centrifuge at 14-16000 x g for 3 min to dry the matrix
• Elution
  1. add 400 μl of W1 buffer into the DFH column
  2. centrifuge at 14-16000 x g for 30 sec. Discard the flow-through and place the DFH column back into the collection tube
  3. add 600 μl of Wash buffer(make sure ethanol was added) into the DFH column
  4. let stand for 1 min at room temp. Centrifuge at 14-16000 x g for 30 sec then discard the flow-through
  5. place the DFH column back in the 2 ml collection tube
  6. centrifuge at 14-16000 x g for 3 min to dry the matrix

Gibson assembly

• Preparation
  • Component

    fragment 1 (? bp)
    fragment 2 (? bp)
    fragment 3 (? bp)
    2X Gibson Assembly master mix	9
    add ddH2O to 18 μl

  • PCR machine set up 50°C for 15 min
  • prepare a 42°C dry bath
  • prepare ice with water
• Calculation
  • Optimized cloning efficiency is 50-100 ng of vector with 2-3 fold molar excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp
• Steps
  1. add the components in following order: ddH₂O, 2X Gibson Assembly master mix, DNA fragments
  2. use PCR machine to incubate for 50°C 15 min
  3. perform the transformation immediately

Ligation

• Preparation

  Aomponent	Amount per Reaction
  vector DNA	0.02 pmol
  insert DNA-1	0.06 pmol
  insert DNA-2	0.06 pmol
  T4 ligase buffer	2 μl
  T4 ligase	1 μl
  add ddH2O to 20 μl

• Steps
  1. add the components in the following order: ddH2O, DNA, T4 DNA ligase buffer, T4 DNA ligase to the eppendorf
  2. stand over 2-4 hour at room temperature
  3. stand overnight at 4°C

Transformation

1. prepare 42°C dry bath, and the ice with water
2. take competent cells out of -80°C and thaw on ice immediately
3. add 2 μl of plasmid and 30 μl competent cell into eppendorf
4. stand on ice for 30 min
5. heat shock for 1 min
6. put the tubes back on ice for 2 min
7. add 1000 μl LB to the eppendorf and incubate at 37°C for 45 min
8. centrifuge at 14-16000 x g for 1 min
9. remove 800 μl of supernatant then resuspend
10. place the transformation onto LB agar plate with selective marker
11. culture for 16-18 hr in the incubator

Competent cell preparation

• DAY 1 - Buffer preparation & bacteria cultivation
  • 0.1M MgCl₂ preparation
  • 0.1M CaCl₂ preparation
  • 85% 100 mM CaCl₂ & 15% glycerol mixture preparation
  • bacteria culture
    • pick a single colony to the 50 ml LB and incubate under 37°C, 200 rpm for 16-20hr
    • two flasks of 125 ml LB are pre-warm under 37°C
• DAY 2
  1. after the 16-20hr cultured, transfer 12.5 ml medium to 125 ml pre-warm LB
  2. the bacteria are cultured to OD value 0.2 (approximately 5hr-6hr)
  3. added 75 ml of pre-warm 37°C LB medium, and culture for another 30 minutes
  Every step should be done on ice
  4. centrifuge 150 ml of the medium at 5000 rpm 10 minutes in 4 °C
  5. discard the supernatant, and resuspend with 37.5 ml MgCl₂
  6. stand for 5 minutes then centrifuge 4000rpm, 10 min. under 4°C
  7. discard the supernatant, and resuspend with 7.5 ml CaCl₂
  8. stand for 20 minutes then centrifuge 4000rpm, 10 min. under 4°C
  9. iscard the supernatant, and resuspend with 1.5 ml CaCl₂+glycerol

Fluorescent intensity and OD value measurement

1. pick a single colony to 5ml LB+CM medium, and culture for 17 hr
2. transfer the cultured medium to LB+CM for final OD value 0.05 and volume 50 ml
3. transfer 100 μl medium to transparent 96 well microplate for OD measurement
4. transfer 100 μl medium to black 96 well microplate for fluorescent intensity measurement
5. repeat step 3 and step 4 hourly until entering the stationary phase